Download On the evolution of Avian infectious bronchitis virus

386
On the evolution of Avian infectious bronchitis virus in
VERO cells
Sobre a evolução do vírus da bronquite infecciosa das galinhas em células VERO
Paulo Eduardo BRANDÃO1,2; Leonardo José RICHTZENHAIN1,2; Laura Yaneth VILLARREAL
Buitrago2,3
Department of Preventive Veterinary Medicine and Animal Health, School of Veterinary Medicine,
University of São Paulo, SP – Brazil
2
Coronavirus Research Group
3
Intervet Schering Plough Animal Health
1
Abstract
Avian infectious bronchitis virus (IBV) (Nidovirales: Coronaviridae) is a chicken Gammacoronavirus with the highest
evolution rate in the genus and, despite the recently reported proofreading activity of its polymerase, intra and interhost diversity is a well documented phenomenon. This study aimed to assess the genetic variation of serial passages of
a variant genotype IBV strain in vitro. Strain CRG-BETA, propagated in chicken embryos, was inoculated in VERO
cells monolayers up to the 4th passage and each passage was monitored with an RT-PCR targeted to the S1 gene (nt 705
to 1094) and an RT-PCR to the protein 5a mRNA. All passages were positive to RT-PCRs to S1 and passages 1 to 3 to
5a mRNA; S1 sequences showed no polymorphism. The finding of IBV mRNA in the cell cultures demonstrates that
the CRG-BETA IBV strain is replicating in the VERO cells and regarding S1 sequence analysis, the lack of nucleotide
mutations shows that CRG-BETA might have reached a fixed status. As a conclusion, different genotypes of IBV present
different evolutionary patterns not only in vivo as previously known, but also in vitro, as described herein.
Keywords: IBV. Spike. Evolution. In vitro.
Resumo
O virus da bronquite infecciosa das galinhas (IBV) (Nidovirales: Coronaviridae) é um Gammacoronavirus com a maior
taxa evolutiva no gênero e, apesar de uma recentemente relatada atividade corretiva de sua polimerase, a diversidade
intra e inter-hospedeiros é um fenômeno bem documentado. Este estudo objetivou avaliar a variação genética após
passagens seriais de uma amostra de IBV variante. A amostra CRG-BETA, propagada em embriões de galinhas, foi
inoculada em monocamadas de células VERO até a quarta passagem e cada passagem foi monitorada com uma RTPCR para a região S1 do gene S (nt 705 a 1094) e uma RT-PCR para o mRNA da proteína 5a do vírus. Todas as passagens
foram positivas para S1 e as passagens 1 a 3 para mRNA 5a; sequências de S1 não apresentaram polimorfismos. O
encontro de mRNA de IBV nos cultivos celulares demonstra que a amostra CRG-BETA está replicando nas células
VERO e, em relação à análise de S1, a ausência de mutações de nucleotídeos demonstra que a amostra CRG-BETA pode
ter atingido um estado fixo. Como conclusão, diferentes genótipos de IBV apresentam diferentes padrões evolutivos não
apenas in vivo, como previamente conhecido, mas também in vitro, como aqui relatado.
Palavras-chave: IBV. Espícula. Evolução. In vitro.
Introduction
Avian infectious bronchitis virus (IBV), currently
named as Avian coronavirus (Nidovirales: Coronaviridae: Coronavirinae: Gammacoronavirus) is a worldwide distributed major pathogen of breeders, broilers
and layers, causing disease of the reproductive, respiratory, urinary and enteric systems of the birds, with
a high attack rate and a major economic impact to the
poultry industry1.
Braz. J. Vet. Res. Anim. Sci., São Paulo, v. 49, n. 5, p. 386-390, 2012
IBV occurs as a wide range of genotypes and serotypes due to the high prevalence of infection and
Correspondence to:
Prof. Dr. Paulo Eduardo Brandão.
Departamento de Medicina Veterinária Preventiva e Saúde Animal.
Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo.
Av. Prof. Dr. Orlando Marques de Paiva, 87, Cidade Universitária.
CEP 05508-270 São Paulo SP - Brasil.
e-mail: [email protected]
Received: 02/03/2011
Approved: 31/10/2012
387
the high mutation rate, while the heterotypic protec-
was inoculated in the monolayers; 1mL of PBS-free
tion amongst different serotypes distantly related is
MEM was inoculated in another flask of cells (mock-
low2. This high evolutionary rate leads thus to a con-
infected).
tinued emergence of nucleotide and amino acids mu-
After an one-hour adsorption at 37ºC, the inocula
tations and eventually to the emergence of novel types
were not discarded and 6mL of MEM plus 2.5% PBS
of the virus .
were added and the monolayers were kept at 37ºC for
3
It’s known that a same initial population of IBV, i.e.,
4 days. After this period, cells were frozen and thaw
a same quasispecies under a neutral network con-
and 1 mL of each previous passage of CRG-BETA or
straint, might go different evolutionary ways depend-
control (mock-infected cells) was used for the next
ing on the type of cell it infects in vivo and that even
passage up to the 4th passage. Cells were observed dai-
a low number of in vitro passages might result in lin-
ly for the presence of cytopathic effect (CPE).
eages divergent form the initial one5.
RNA extraction
4
A typical Brazilian IBV lineage has been described
6,7
All RNA extractions were carried out with TRIzol
with at least three sub lineages consistently diver-
Reagent™ (Invitrogen, Carlsbad, CA, USA) according
gent from the archetypical strains already described.
to manufacturer`s instructions.
3
Nonetheless, there’s no data on the effect of serial passages of these viruses neither in vivo nor in vitro.
As the knowledge on the molecular behavior of IBV
is paramount for the knowledge on vaccine strain stability, the aim of this investigation was to assess the
divergence of a Brazilian strain of IBV in cell culture
based on a sequence of the spike envelope glycoprotein (S) gene, a major marker for diversity in this virus
species.
Reverse-transcription polymerase chain reaction
(RT-PCR) to the 5a protein mRNA
For the detection of viral transcription activity, each
passage (CRG-BETA and control) was monitored for
the presence of mRNA of the non-structural protein
5a of IBV.
Complementary DNA was synthesized with M-MLV
Reverse Transcriptase™ (Invitrogen, Carlsbad, CA,
USA) according to manufacturer’s instructions using
the anti-sense primer M41N-AS 5’ CACACGTTAA-
Material and Method
GATGTGTTTTGGTCC 3’, complementary to positions 164 to 188 of the 5a gene sequences AY392047.1,
Virus strains
The strain CRG-BETA isolated in chicken embryos
X03448.1, DQ834384.1 and AY851295.1.
(4 passage) and typed as Brazilian genotype was used
PCR was carried out with Platinum Taq DNA Poly-
for the in vitro inoculations. The H120 vaccine strain
merase™ (Invitrogen, Carlsbad, CA, USA) accord-
(Massachusetts serotype) was used as a positive con-
ing to manufacturer`s instructions and an annealing
trol in the RT-PCRs (DEPC-treated water was used as
temperature of 48ºC with the antisense (M41N-AS)
the negative control).
and sense (Niña-S 5’ TCTATCACACTAGCCTT-
Passages in VERO cells
GCGCTAGA 3’, nucleotides 25 to 49 of sequences
th
Forty-eight hour-old confluent VERO cells grown
AY392047.1, X03448.1, DQ834384.1 and AY851295.1,
in 25cm2 flasks with MEM (minimal essential medi-
correspondent to the leader sequence). After agarose
um) with 10% phoetal bovine serum (PBS) were used
gel electrophoresis strained with ethidium bromide,
for all inoculations. The growth medium was discard-
reactions with the predicted 164bp amplicons were
ed and 1mL of CRG-BETA (4 passage in embryos)
considered as positives.
th
Braz. J. Vet. Res. Anim. Sci., São Paulo, v. 49, n. 5, p. 386-390, 2012
388
RT-PCR for the S1 subunit of the spike glycoprotein
gene
RT-PCR for NewCastle Disease Virus (NDV)
All passages, including the mock-infected controls,
Each passage (CRG-BETA and control) was sub-
were monitored for NDV as described by Tiwari et al.10.
mitted to an RT-PCR targeted to the amplification
of a 390bp fragment of the region coding for the S1
Results
subunit of the spike glycoprotein (nt 705 to 1094) as
described by Worthington, Currie and Jones8 using
Passages in VERO cells
M-MLV Reverse Transcriptase™ (Invitrogen, Carlsbad, CA, USA) according to manufacturer’s instructions and Random Primers ™ (Invitrogen, Carlsbad,
CA, USA) for the cDNA synthesis and Platinum Taq
DNA Polymerase™ (Invitrogen, Carlsbad, CA, USA)
according to manufacturer`s instructions for the first
round and nested PCRs.
passage (Figure 1B), while no alterations were detected in the mock-infected monolayers (Figure 1A). By
the 4th passage, no CPE was detected.
RT-PCRs to the 5a protein mRNA and S1 subunit
Passages 1 to 4 and 1 to 3 of CRG-BETA in VERO
cells resulted positive for the RT-PCR to the S1 region
and to the 5a mRNA, respectively, while no amplifica-
DNA sequencing
S1 amplicons were purified from agarose gels using
Ilustra™ GFX PCR DNA and Gel Band Purification kit
(GE Healthcare, USA), bi-directional DNA sequencing was carried out with BigDye 3.1™ (Applied Biosystems Cidade, País) and sequences resolved in an ABI3130™ Automatic Sequences (Applied Biosystems™).
Chromatograms were analyzed with Phred online
(http://asparagin.cenargen.embrapa.br/phph/)
Syncytia were observed from the first to the third
and
tions were detected in the mock-infected cells.
DNA sequencing
The partial sequences of the S1 were obtained
for passages 2 to 4 and from the original CRG-BETA strain in embryos. No mutations were detected
amongst the four sequences in the 316nt alignment
obtained (Figure 2).
RT-PCR for (NDV)
only positions with scores higher than 20 were used.
All VERO cells passages of CRG-BETA were found
Sequences were assembled with Cap-Contig and
aligned with CLUSTAL/W using Bioedit v. 5.0.99.
positive for NDV. No NDV was detected in the mockinfected monolayers.
Figure 1 - VERO cells monolayers 100 hours post-infection with mock inoculum (A) or CRG-BETA IBV (B) at the 3rd
passage; 100x magnification
Braz. J. Vet. Res. Anim. Sci., São Paulo, v. 49, n. 5, p. 386-390, 2012
389
Figure 2 - Partial S1 subunit sequences of IBV strain CRG-BETA ate the second, third and fourth passages in VERO cells
and the original strain in embryos
Discussion
In this study, a Brazilian genotype IBV strain was
serially passaged in VERO cells and no mutations in
a region of the S1 subunit nucleotide sequences were
detected.
The detection of mRNA transcription, achieved
in this study with an RT-PCR to the 5a gene mRNA,
clearly demonstrates that the CRG-BETA strain is
replicating in the cells and that the RNA detected is
not genomic or inoculum-derived.
In association with the appearance of syncytia, a
typical IBV cytopathic effect in cell culture5, these results demonstrate that, at least up to the third passage,
this strain was able to replicate and to spread from cell
to cell with the methods employed herein.
Nonetheless, is noteworthy that passage 4 produced
no CPE or mRNA was detected. One can hypoth-
population might have decrease to a low number at
the 4th passage, entering in a bottleneck phase. A higher number of passages is necessary to verify this effect
and the quantization of mRNA transcripts by qPCR
would also shed some light on that.
The stability of the S1 region analyzed is also an interesting finding, as others5 have described a less mutation-robust spike gene for IBV passages in VERO
cells using the Beaudette strain as an inoculum.
The Beaudette strain belongs to the Massachusetts
serotype of IBV, while CRG-BETA is a Brazilian genotype one, with a maximum reported nucleotide identity of 79.3% for this S1 region amongst these types3.
As the spike protein plays a major role in receptorbinding11, it can be speculated that this difference in
S might have played a role in the different evolutionary pathways specifically endowing CRG-BETA with
a more mutation-robust S when replicating in VERO
esize that CRG-BETA was present in a high titer in
cells. Anyhow, again this speculation must be taken
the original, embryo-derived, strain and was thus able
with care, as further passages and large S sequences
to efficiently replicate in the first passages due to the
would be necessary to assess its consistency. Also, as
high IBV population number present in the inocu-
non-structural proteins might play an important role
lum, but, arguably due to intense selection, the IBV
in pathogenesis2, differences in these proteins might
Braz. J. Vet. Res. Anim. Sci., São Paulo, v. 49, n. 5, p. 386-390, 2012
390
also have accounted for a different host-virus rela-
which might serve as a model not only for the assess-
tionship in the cytoplasmic environment.
ment of vaccine strain stability, but also for a deeper
Finally, the presence of NDV must also be accommodated in the hypothesis for the evolutionary patterns described herein. It’s known that co-infecting
viruses, such as IBV and Avian metapneumovirus
might interfere with each other’s replication in vivo
due to receptor competition12 and thus NDV might
have outreplicated IBV CRG-BETA in the monolayers
and led it to extinction.
understanding of co-evolution co-infecting viruses in
vivo and in vitro.
Acknowledgements
The authors are grateful to FAPESP (Fundação
de Amparo à Pesquisa do Estado de São Paulo) and
CNPq (Conselho Nacional de Desenvolvimento
As a conclusion, an exclusive evolutionary pattern
Científico e Tecnológico), grants # 2008/58649-4 and
of a Brazilian genotype IBV strain has been detected
300513/2010-0, respectively, for the financial support.
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